Synthetic mammalian neuromuscular junction and method of making

ABSTRACT

A method for forming neuromuscular junctions includes forming functional neuromuscular junctions between motoneurons and muscle cells by co-culturing one or more human motoneurons and one or more human muscle cells in a substantially serum-free medium. A synthetic mammalian neuromuscular junction includes a human motoneuron functionally linked to a human muscle cell in a substantially serum-free medium. An artificial substrate may be used to support the one or more neuromuscular junctions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application of InternationalApplication No. PCT/US2011/035585, filed May 6, 2011, which claimspriority to U.S. Patent Application No. 61/332,003, filed May 6, 2010,and is a continuation-in-part of U.S. patent application Ser. No.12/765,996, filed Apr. 23, 2010, which in turn claims priority to U.S.Patent Application No. 61/171,958, filed on Apr. 23, 2009, the entirecontents of which are incorporated herein fully by this reference.

STATEMENT OF GOVERNMENT INTEREST

The invention claimed herein was made with at least partial support fromthe U.S. Government under National Institutes of Health grantR01NS050452. Accordingly, the U.S. Government may have certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates to the field of cell culture, and, moreparticularly, to formation of neuromuscular junctions.

BACKGROUND OF THE INVENTION

Neuromuscular junction (“NMJ”) formation is a complex process thatdepends on many variables. Unfortunately, current techniques forproducing NMJs suffer from one or more drawbacks which hinder theirreproducibility and utility.

For centuries, animals and animal-derived tissues have been the majortools for understanding biological systems, human diseases, developingtherapeutic strategies and screening drugs. However, translating animaldata to clinical applications has been problematic, leading to fewerdrugs being approved and an increasing cost in the drug discoveryprocess (58). While some functional in vitro systems composed of humancells has been reported for liver (59), skin (60,61) and cardiomyocytes(62,63), no system composed of human cells has been reported forneuronal systems. Systems based on functional NMJs are of particularinterest due to the fact that NMJs represents a synapse-based model thatwould be clinically applicable to spinal cord injury andmotoneuron-related diseases such as Amyotrophic lateral sclerosis(“ALS”) (64), spinal muscle atrophy (65) and muscular dystrophy (66). Anin vitro (1) co-culture system composed of human motoneurons andskeletal muscle would be useful for studies ranging from understandingNMJ synaptogenesis, target generation for NMJ related diseases,screening therapeutic candidates and conducting drug toxicologyevaluation. The advantages of human-based in vitro systems compared toin vivo systems reside in that they are much simpler and therefore easyto manipulate any factors, to dissect the mechanisms or pathways and toanalyze the results.

One technique for forming NMJs in vitro to use a motoneuron(“MN”)-muscle cell co-culture. MN-muscle co-cultures have been describedin Xenopus (1, 2), chick (3-5), mouse (6, 7) and rat (8, 9), as well asin cross-species investigations between mouse MN-chick muscle (7, 10),human stem cell-derived MNs-myotubes from C2C12 cells (11). One drawbackto these in vitro MN-muscle co-culture systems is that they use serumcontaining media and a biological substrate (3-5,8,9). Since the serumcontaining medium contains many unknown components and because of thetechnical difficulties in creating reproducible biological substrates,these examples have led to undesired culture variability, making itextremely difficult, if not impossible, to ascertain the minimum factorsrequired for recreating or maintaining the NMJ in vitro.

Due to the variability inherent with serum containing media (12),serum-free NMJ formation systems have been developed. NMJ formation inserum-free systems has been demonstrated using rat cells (13). Also,cross species NMJ formation between human MN and rat muscle (14) hasbeen demonstrated. These in vitro systems comprised of animal-derivedcomponents have provided the scientific community with readily availablemodels for understanding NMJ synaptogenesis and NMJ-related diseases,however, in order to understand NMJ formation in all human cells, theresults from these systems must be extrapolated, which can bedisadvantageous for clinical applications among others.

A major hurdle in building in vitro biological systems using humancomponents is limitations related to tissue source. However, recentdevelopments in stem cell biology provide an avenue to, not only have anunlimited supply of human cells for tissues, but also to provide geneticdiversity in the systems. Cloned human skeletal muscle satellite cellshave been used for studying NMJs in vitro by combining them with ratspinal explants or dissociated MN in serum-containing systems (15-19).MNs derived from human embryonic stem cells (“hESC”) (11) and humanfetal spinal cord stem cells (“hSCSC”) (20) have been studied. NMJformation has been (2) demonstrated between hESCs and C2C12 cells in aserum based system (11), as well as between hSCSCs and rat myotubesderived from embryonic skeletal muscles in a defined serum-free system(14). However, no human based in vitro NMJ system, in which both MNs andmyotubes were derived from stem cells presently exists. Accordingly,there is a need in the art for a human based system for NMJ formationthat does not suffer from one or more of the above described drawbacks.

SUMMARY

Certain embodiments of the invention are directed to methods thatsatisfy the need for a human based NMJ formation system. In oneexemplary embodiment, the method comprises forming functionalneuromuscular junctions between motoneurons and muscle cells byco-culturing one or more human motoneurons and one or more human musclecells in a substantially serum-free medium.

In another embodiment the method comprises suspending human skeletalmuscle cells in a serum-free medium; suspending human motoneuronsderived from human spinal cord stem cells in the serum-free medium;plating the suspended muscle cells and the suspended motoneurons onto anartificial carrier; and monitoring for formation of functionalneuromuscular junctions.

Other embodiments of the invention are directed to neuromuscularjunctions that satisfy this need. In one example, the embodiment isdirected to synthetic mammalian neuromuscular junction comprising ahuman motoneuron functionally linked to a human muscle cell in asubstantially serum-free medium. The human motoneuron can befunctionally linked to the human muscle cell on an artificial surface. Apreferred artificial surface has a silicon based monolayer substratedeposited thereon, which may, if desired, be deposited in apredetermined pattern.

In certain embodiments, the substantially serum-free medium iscompletely serum free. Some examples of the substantially serum-freemedium comprise at least one synaptogenesis promoting component and oneor more trophic factors. NbActiv4 can be added to the serum-free medium.In a preferred embodiment, the medium comprises the components in Table1.

Preferrably, but not necessarily, the human motoneuron cells are derivedfrom human spinal cord stem cells and the human muscle cells are derivedfrom human skeletal muscle stem cells.

Some embodiments can include a synthetic substrate adapted to support atleast one neuromuscular junction thereon. The synthetic substrate ispreferably silicon based and more preferably is DETA. The syntheticsubstrate may be deposited on a support surface in a predeterminedpattern if desired. The synthetic substrate may be coated on a carrier.

These and other objects, aspects, and advantages of the presentinvention will be better appreciated in view of the drawings andfollowing detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of phase contrast microscopy images showing humanskeletal muscle cells (hSKMs) and human motoneurons (hMNs) in cultureand co-culture, according to an embodiment of the present invention; A.Depicts myocytes that were allowed to grow to confluency beforedifferentiation was induced; B. Depicts, multi-nuclei myotubes that wereinduced during differentiation; C. Depicts both myotubes and neuronsthat survived in the coculture; D. Illustrates connections betweenneurons and myotubes in the co-culture (indicated by arrows); E. Showsthe striation of myotube as indicated by the yellow arrow; F.Illustrates a neuron with MN morphology sends out long axons towards astriated myotube as indicated by the red arrow;

FIG. 2 is a set of microscopy images showing that after one week ofco-culture the morphology of hMNs and hSKM myofibers were well definedand easily distinguishable; A & B. Shows a neuron sent an axon towards amyotube and branched at the contacts with myotubes as indicated by thearrows; C. Depicts co-immunostaining of MHC (myosin heavy chain) with βIII Tubulin in a 19 day co-culture;

FIG. 3 is a set of microscopy images depicting synaptophysin-positiveterminals co-localized with AchR clusters; potential synaptic sites(yellow arrows) demonstrated by co-localization of nerve terminals(indicated by synaptophysin) and AchR (indicated by BTX488), in a day 15coculture—Scale Bars A. 20×. B. 40×; and

FIG. 4 is a representative graph of voltage-clamp and current-clamprecordings for the MNs and myotubes; A&B. Representative Voltage clamp(A) and Current clamp (B) trace recording on myotubes in the co-culture;C&D. Representative Voltage clamp (C) and Current clamp (D) traceRecording on motoneurons in the co-culture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the Summary of the Invention above and in the Detailed Description ofthe Invention and in the accompanying drawings, reference is made toparticular features (including method steps) of the invention. It is tobe understood that the disclosure of the invention in this specificationincludes all possible combinations of such particular features. Forexample, where a particular feature is disclosed in the context of aparticular aspect or embodiment of the invention, that feature can alsobe used, to the extent possible, in combination with and/or in thecontext of other particular aspects and embodiments of the invention,and in the invention generally.

The term “comprises” is used herein to mean that other ingredients,features, steps, etc. are optionally present. When reference is madeherein to a method comprising two or more defined steps, the steps canbe carried in any order or simultaneously (except where the contextexcludes that possibility), and the method can include one or more stepswhich are carried out before any of the defined steps, between two ofthe defined steps, or after all of the defined steps (except where thecontext excludes that possibility).

In this section, the present invention will be described more fully withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein.

According to an embodiment of the invention, an in vitro system forforming NMJs between human cells is provided. The system comprises an invitro co-culture adapted to allow NMJs to form between human neurons andhuman muscle cells in a defined environment. The defined environment ispreferably achieved by utilizing a co-culture medium in which theingredients and quantities of those ingredients are known. In apreferred embodiment, the medium contains no serum. The co-culture canalso be prepared on substrate that has a defined surface, such as byassembling a synthetic material onto an underlying surface for example.In some cases, the synthetic material can be assembled on the underlyingsurface according to a desired pattern.

In vitro NMJ co-culture systems containing human cellular componentshave been reported previously. Human stem cell-derived motoneurons canform NMJs when co-cultured with C2C12 cells (11) from co-cultures ofembryonic rat spinal explants with human SKM stem cell-derived myofibers(17, 32), but in serum containing media. One study also investigatedhuman stem cell derived motoneuron innervations for rat embryonic SKM ina defined system (14). These systems were employed to study thefunctional integrity of motoneurons differentiated from human stem cells(11, 14), to investigate the functional maturation process of SKMsderived from human SKM stem cells (33, 34), the mechanisms of NMJformation on human SKMs (15-19, 30-36), and the pathogenesis of somespinal muscular diseases (37).

This disclosure reports the first human-based in vitro NMJ system whichsupports the differentiation of human stem cell derived motoneurons andSKMs and provides for functional NMJ formation. The system developed inthis study, by the co-culture of human stem cell-derived motoneurons andSKMs, provides a system closer to the human condition that is capable ofaddressing the above described drawbacks, as well as neurological and/ormuscular disease modeling, drug discovery and regenerative medicine.

In an exemplary embodiment, the human neurons are MNs differentiatedfrom human spinal cord stem cells and the muscle cells are humanskeletal muscle stem cells.

By way of example, a suitable co-culture medium that can be used in thehuman NMJ formation system is comprised of the ingredients provided inTable 1. The scope of the invention is not limited only to theseingredients, nor is it required that every one of the ingredients beused in every embodiment. Ingredients may be added to or taken away fromTable 1 without falling outside the scope of the invention. Thecombination of NEUROBASAL™ medium, B27, Glutamax, GDNF, BDNF, Shh, RA,IGF-1, cAMP, CNTF, NT-3, NT-4, Vitronectin and Laminin has been found tobe able to support the growth, differentiation, and long-term survivalof MNs derived from human stem cells (11, 14). Laminins are importantcomponents of the extracellular matrix that facilitates synaptogenesis(1). Specifically, β2 laminins are concentrated at synaptic sites andare useful for their postnatal maturation (57). The addition of the G5supplement to the co-culture medium has been found to significantlyenhance myocyte proliferation. However, the continuous presence of thesetrophic factors, including BDNF, GDNF, NT-3, NT-4 and cAMP, was found tosignificantly down regulate agrin deposition along the neurites and atnerve-muscle contacts, thus preventing synaptogenesis (2).

In a preferred preparation of NMJs, the trophic factors were graduallywithdrawn and the culture was fed using only NbActiv4 media. TheNbActiv4 media formula was generated by adding three ingredients,cholesterol, estrogen, and creatine to media containing NEUROBASAL™, B27and Glutamax (53). There is evidence that the addition of theseingredients can significantly promote synaptogenesis (53-56). Therefore,the co-culture was first plated in the co-culture medium to ensure thesurvival and growth of MNs and myocytes, followed by the gradualwithdrawal of these factors which enabled the reciprocal inductionbetween the MNs and myotubes that naturally occurs in development.

Advantageously, the defined co-culture medium delineates the basis forthe essential components during NMJ formation, and provides a basicsystem for dissecting the individual factors, for investigating theunderlying mechanisms, and later for treatment of diseases related tothe cellular components of NMJs.

A preferred substrate is trimethoxysilylpropyldiethylenetri-amine(“DETA”), which can be coated onto a carrier or surface such as a glasscover slip for example. In the working examples discussed below, DETAwas coated on a glass surface to form a self-assembled monolayer. DETAhas previously been shown to support neuronal (26), skeletal muscle(27), endothelial (38), and cardiac cell growth (39), and has been usedin creating high-resolution, in vitro patterned circuits of embryonichippocampus neurons (40). Moreover, DETA substrates have been shown topromote guided axonal growth and direct axonal and dendritic processextension at the level of a single neuron (41). Therefore, thesuccessful formation of NMJ on this substrate implies that someco-cultures of the invention can be patterned at high resolution tostudy engineered in vitro NMJs. Especially, this surface modificationtechnique can be used for guiding specific NMJ formation.

Functional in vitro systems composed of human cells in a defined,serum-free system, especially those reproducing fundamental neurologicalprocesses, will be a significant component in transforming currentmethods of drug discovery and toxicology. The utilization of neuronalsystems derived from stem cells enables a process that can begenetically diverse, yet source reproducible. The use of a defined,serum-free system also enables the integration into the next generationof high-content and ultimately high-throughput screening technologies.

Accordingly, embodiments of the invention have many advantages. Some,but not all, of those advantages are listed here. Not all of theseadvantages are required by all embodiments of the invention. In summary,embodiments of the invention provide the first pure human based NMJ invitro culture system. This human cell-based system bridges the gapbetween findings from animals and their clinical applications. The stemcell origin for both motoneurons and skeletal muscles enables theformation of these cultures in large quantities which can be importantfor high throughput drug screening. The serum-free medium allows thissystem to be highly re-producible and easy to manipulate. Thepatternable surface gives the power to the system to be engineered intoneural circuits. These attributes indicate that this system willfacilitate not only the studies concerning human NMJ development andregulation, both in vitro and in vivo, but also the research fieldstargeting NMJ-related diseases and treatment, such as by developing highinformation content drug screen systems and test beds in pre-clinicalstudies.

In the following section, we describe several working examples in whichan exemplary human NMJ model system embodiment was characterized bymorphology, immunocytochemistry, and electrophysiology. Further, NMJformation was demonstrated by immunocytochemistry and videography.

WORKING EXAMPLES DETA Surface Modification

Glass coverslips (6661F52, 22×22 mm No. 1; Thomas Scientific,Swedesboro, N.J., USA) were cleaned using HCl/methanol (1:1) for atleast 2 hours, rinsed with water, soaked in concentrated H2SO4 for atleast 2 hours and rinsed with water. Coverslips were boiled in nanopurewater and then oven dried. The trimethoxysilylpropyldiethylenetri-amine(DETA, T2910KG; United Chemical Technologies Inc., Bristol, Pa., USA)film was formed by the reaction of cleaned surfaces with a 0.1% (v/v)mixture of the organosilane in freshly distilled toluene (T2904; Fisher,Suwanne, Ga., USA). The DETA coated coverslips were heated to ˜80° C.,then cooled to room temperature (RT), rinsed with toluene, reheated toapproximately the same temperature, and then cured for at least 2 hoursat 110 C. Surfaces were characterized by contact angle and X-rayphotoelectron 5 spectroscopy as described previously (26, 42, 43).

Co-Culture of Human MNs and Human Skeletal Muscle Stem Cells

Materials and Methods.

The human spinal cord stem cell line was isolated and established asdescribed in (44-46). MNs were differentiated from this cell line asdescribed in (20). Briefly, ˜1×106 hSCSCs were plated in one 60 mmparanox cell culture dish (Nunc, Cat #174888) and differentiated 4 daysin the priming media followed by 6 days in differentiation media. Thecomposition of the priming media and differentiation media weredescribed in (20).

Human skeletal muscle stem cells (hSKM SCs) were isolated, proliferatedand differentiated as described in (47). Briefly, primary human skeletalmuscle cells isolated by needle biopsy (48) were expanded in myoblastgrowth medium (MGM; SkGM (Cambrex Bio Science, Walkersville, Md.) plus15% (v/v) fetal bovine serum). Biopsies were performed on adultvolunteers according to procedures approved by the InstitutionalClinical Review Board of the Miriam Hospital. Cell preparations onaverage were 70% myogenic based on desmin positive staining (49).Myoblast fusion into postmitotic myofibers was induced by incubation indifferentiation medium (high-glucose DMEM (Invitrogen, Carlsbad, Calif.)supplemented with insulin (10 μg/ml), bovine serum albumin (50 μg/ml),epidermal growth factor (10 ng/ml) and gentamicin (50 μg/ml)). For eachculture, hSKM SCs were plated on DETA coverslips at a density of 20cells/mm2 in hSKM Growth Medium (Lanza, Cat# CC-3160), fed every 2 daysby changing the whole medium. On day 7, myoblast fusion was induced byswitching to differentiation medium. The cells were fed every 2 days bychanging half of the medium. On day 7 after differentiation,differentiated hSCs were harvested and plated on top of these inducedmyotubes in a density of 200 cells/mm2, and the medium was changed toco-culture medium (Table 1). Two days later, the medium was fed byco-culture medium (without G5) by changing half of the medium. Afteranother two days and thereafter, the cultures were fed by NbActiv4(Brain Bits) by changing half of the medium.

Discussion.

The procedure of co-culturing motoneurons and skeletal muscles (SKMs)was described in detail in the Material and Methods. Briefly, human SKMstem cells were allowed to grow to confluence before induction ofdifferentiation (FIG. 1A). After switching to differentiation media, thefusion of myocytes was initiated. Multi-nuclei myotubes formed graduallyand were prevalent from day 4 in the culture (FIG. 1B). Differentiatedhuman motoneurons (hMNs) were cultured as in Guo et al. (20) and wereplated on the top of the differentiated myotubes and the medium wasswitched to a co-culture medium at this time. Both hSKMs and hMNssurvived well in the co-culture media (FIG. 1C). Also, thedifferentiation of both hMNs and hSKMs were evident after one week. ThehMNs were easily identifiable in the co-culture and sent out axonseither along or ending at the myotubes (FIGS. 1D, E). In addition, alarge number of human myotubes exhibited striated band patterns (FIGS.1E, F). This characteristic A & I band patterning is caused bydifferential light diffraction due to the organization of myofibrialproteins forming sarcomeres within the myotubes, and is observed withmature in vivo muscle fibers (21, 22). The striated patterns indicatedthe formation of the basic contractile apparatus for skeletal muscle,implying that these myofibers were structurally and functionally mature.

Multiple plating conditions were examined to determine the optimalculturing procedures. When plating differentiated motoneurons on top ofSKMs before extensive myotube formation the myocyte fusion proceededsub-optimally when switched to the co-culture medium, and with theformation of a minimal number of multi-nuclei myotubes. The viabilityand morphological differentiation of the replated motoneurons was alsopoor, overall indicating that the co-culture media is not favorable forthe fusion of human myocytes, and the successful replating ofmotoneurons required the pre-differentiation of the SKMs. This isreasonable considering that muscle cells release the neurotrophins BDNF,GDNF and NT-3/4 to support MN survival and attract neurite outgrowth ofmotoneurons during development (23-25).

Another observation was that when the co-culture was fed for four dayswith co-culture medium containing G5, undesired proliferation fromundifferentiated stem cells was observed. When G5 was removed from theco-culture medium completely, however, the replated motoneurons survivedpoorly. To mediate this complication, the G5 was kept in the originalplating medium for the co-culture and then was gradually removed aftertwo days.

Immunocytochemistry and Microscopy

Materials and Methods.

Cells on DETA coverslips were fixed in freshly prepared 4%paraformaldehyde for 15 min. For the co-stainings with BTX-488, cultureswere incubated with BTX-488 (Invitrogen, Cat# B13422) at 1×10-8M for 1hr in the 37° C. incubator before fixation. Cells were then washed twicein Phosphate Buffered Saline (PBS) (pH 7.2, w/o Mg2+, Ca2+) for 10 mineach at room temperature, and permeabilized with 0.1% triton X-100/PBSfor 15 min. Nonspecific binding sites were blocked in Blocking Buffer(5% Donkey serum plus 0.5% BSA in PBS) for 45 min at room temperature.Cells were then incubated with primary antibodies overnight at 4° C.After being washed with PBS 3×10 min, the cells were incubated withsecondary antibodies for 2.5 hours at room temperature. The cells werethen washed with PBS 3×10 min and mounted with Vectorshield with4′-6-Diamidino-2-Phenylindole (dapi) (Vector laboratories, Inc.).Primary antibodies used in this study include: Rabbit-anti-β III Tubulin(Sigma, 1:1500), Mouse-anti-synaptophysin (Antibodies Inc., 1:100). Themonoclonal antibody against muscle heavy chain (MHC, F1.625, 1:10) wasobtained from the Developmental Studies Hybridoma Bank which is underthe auspices of the NICHD and maintained by the University of Iowa.Secondary antibodies include: Donkey-anti-Mouse-488 (Invitrogen, 1:250)and Donkey-anti-Rabbit-594 (Invitrogen, 1:250). All antibodies werediluted in Blocking Buffer.

Discussion.

After one week of co-culture, the morphology of hMNs and hSKM myofiberswere well defined and easily distinguishable, and it was observed thatthe hMNs axons terminated and even branched at the contact withmyofibers (FIGS. 2A, B). Utilizing immunocytochemical analysis withβ-III Tubulin for neurons and muscle heavy chain (MHC) for themyofibers, the details of these contacts were confirmed. In theco-culture system the nerve endings branched in the vicinity of myotubeand the terminals wrapped around the myotubes as shown in FIG. 2C. Thisimage reproduces previous findings during NMJ formation which indicatedthat synaptogenesis is a dynamic process directly correlated to theactive branching and remodeling of axon terminal arbors (28, 29). Thepotential for NMJs in the culture were further analyzed by thecoimmunostaining of BTX-488 (a-bungarotoxin, Alexa Fluor® 488 conjugate)and synaptophysin, a synaptic vesicle protein. As shown in FIG. 3,synaptophysinpositive terminals co-localized with AchR clusters, astrong indication for NMJ formation.

Electrophysiological Properties

Materials and Methods.

Electrophysiological properties of spinal cord stem cell-derivedmotoneurons and human myotubes were investigated after ˜10 days in thecoculture using whole-cell patch-clamp recording techniques (26). Therecordings were performed in a recording chamber located on the stage ofa Zeiss Axioscope 2FS Plus upright microscope (50).

Motoneurons were identified visually under an infraredDICvideomicroscope. The largest multipolar or round cells (15-25 μmdiam) with bright illuminance in the culture were tentatively identifiedas motoneurons (51, 52). Patch pipettes with a resistance of 6-10 MOwere made from borosilicate glass (BF 150-86-10; Sutter, Novato, Calif.)with a Sutter P97 pipette puller (Sutter Instrument Company).

Current-clamp and voltage-clamp recordings were made utilizing aMulticlamp 700A amplifier (Axon, Union City, Calif.). The pipette(intracellular) solution contained (in mM) K-gluconate 140, MgCl2 2,Na2ATP 2, Phosphocreatine 5, Phosphocreatine kinase 2.4 mg, Hepes 10; pH7.2. After the formation of a gigaohm seal and the membrane puncture,the cell capacitance was compensated. The series resistance wastypically <23 MO, and it was compensated >60% using the amplifiercircuitry. Signals were filtered at 3 kHz and sampled at 20 k Hz using aDigidata 1322A interface (Axon instrument).

Data recording and analysis were performed with pClamp8 software (Axoninstrument). Membrane potentials were corrected by subtraction of a 15mV tip potential, which was calculated using Axon's pClamp8 program.Membrane resistance and capacitance were calculated using 50 ms voltagesteps from −85 to −95 mV without any whole-cell or series resistancecompensation. The resting membrane potential and depolarization-evokedaction potentials were recorded in current-clamp mode.Depolarization-evoked inward and outward currents were examined involtage-clamp mode.

Monitoring the contraction of human skeletal muscles in the co-cultureand the determination of the effect of (+)-tubocurarine chloridepentahydrate (dtubocurarine or curare) on the NMJs by video recording

Discussion.

The electrophysiological properties of the MNs and myotubes in theco-culture were evaluated using voltage and current clamp recordings foreach cellular component. Representative voltage-clamp and current-clamprecordings for the MNs and myotubes are shown in FIG. 4. The electricalproperties of MNs in the co-culture system, such as membrane resistance,resting membrane potential, Na+/K+ current amplitude, the ability torepetitively fire and the amplitude of action potential (AP), werecomparable to results described previously (20, 26). The electricalproperties for the myotubes were also comparable to previously publishedresults (27).

Videography of NMJ Formation

Materials and Methods.

Functional NMJ formation was investigated in the co-culture system 1˜2weeks after MN plating utilizing video recordings. In each experiment,the coverslip in a 6-well plate was maintained in NBActiv media in atime lapse chamber (37C, 5% CO2) located on the stage of a ZeissAxiovert microscope 200. The videos were recorded by a Hamamatsu digitalcamera (Model C848405G) at a frame rate of 8 frames/sec using WindowsMovie Maker software. For the experiments with curare, 100 μl of theNicotinic cholinergic antagonist, (+)tubocurarine chloride pentahydrate(also known as curare, cat. no. 93750, Sigma) (stock 250 μM, final 8 μM)was applied to the bath solution to block theacetylcholine receptorspresent in the NMJs. This concentration was chosen based on previousstudy (43).

Discussion.

The results obtained from several of the videos will now be discussed.In Video 1 (10 min), during the first 6 min, the myotube contracted inpulses which was recurrent approximately every 1-2 min. The contractionthen stopped after this time period. In Video 2 (15 min), thecontraction pulses of three spots were observed to intermittentlycontract. In Video 3 (15 min), myotube contraction was recorded for 7min. The addition of Curare (5 μM) silenced the contraction. In Video 4(22 min), myotube contraction was recorded for 11 min. The addition ofCurare (5 μM) silenced the contraction.

Numerous muscle contractions in the absence of any stimulus could beobserved in the co-culture approximately one week after the introductionof hMNs this was based on observations from more than 15 coverslips outof 5 independent platings. All Videograph experiments were conducted ina system in which the cultures were kept in the chamber at 5% CO₂ 37 Cconditions. They would stop in ambient condition soon after (withinminutes) being taken out of the incubator. Contractions with a constantrhythm in the hMN-hSKM co-cultures were present as in Video 2, as wellas contractions exhibiting a constant pattern of intermittent pulses asdemonstrated in Video 1. Video 1 indicated a myotube contracting inpulses with an approximately 1-2 min interval. These recurrentcontractions lasted for 6 min and stopped thereafter. In Video 2,recorded from another site, three other areas were shown to contract onand off intermittently. The contractions in the hMN-hSKM co-culture weretested by tubocurarine for their source of initiation. As in Video 3 andVideo 4, the contractions ceased after the application of 100 ul oftubocurarine (200 uM) to the culture (final 5 μM), confirming theneuronal initiation of these contractions and the formation of NMJs.This experiment with curare was repeated 4 times with the same result.

These muscle contractions revealed a few differences compared to theco-cultures of hMN with rat embryonic SKMs (14). Although studies fromco-cultures of fetal rat spinal cord explant with human myoblastsconsistently indicated that hSKMs didn't have spontaneous contractionand any contraction in the co-culture was an indication of innervations(16-19, 30, 31).

The present invention has been described hereinabove with reference tothe accompanying drawings, in which preferred embodiments of theinvention are shown. Unless otherwise defined, all technical andscientific terms used herein are intended to have the same meaning ascommonly understood in the art to which this invention pertains and atthe time of its filing. Although various methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed. However, the skilled should understand that the methods andmaterials used and described are examples and may not be the only onessuitable for use in the invention.

Moreover, it should also be understood that any temperature, weight,volume, time interval, pH, salinity, molarity or molality, range,concentration and any other measurements, quantities or numericalfigures expressed herein are intended to be approximate and not an exactor critical figure unless expressly stated to the contrary.

Further, any publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety as if they were part of this specification. However, in case ofconflict, the present specification, including any definitions, willcontrol. In addition, as noted above, materials, methods and examplesgiven are illustrative in nature only and not intended to be limiting.

Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough, complete, and will fully convey thescope of the invention to those skilled in the art. Therefore, in thespecification set forth above there have been disclosed typicalpreferred embodiments of the invention, and although specific terms areemployed, the terms are used in a descriptive sense only and not forpurposes of limitation. The invention has been described in some detail,but it will be apparent that various modifications and changes can bemade within the spirit and scope of the invention as described in theforegoing specification and as defined in the appended claims.

Any element in a claim that does not explicitly state “means for”performing a specified function, or “step for” performing a specifiedfunction, is not to be interpreted as a “means” or “step” clause asspecified in 35 U.S.C. §112, ¶ 6. In particular, the use of “step of” inthe claims herein is not intended to invoke the provisions of 35 U.S.C.§112, ¶ 6.

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TABLE 1 Composition of Enriched Co-culture Media. Catalog Component FullName Concentration Company Number NEUROBASAL ™/ Invitrogen 10888/21103NEUROBASAL ™ A B27 (50X) 1X Invitrogen 17504-044 Glutamax (100X) 1XInvitrogen 35050 GDNF Glial-derived  10 ng/ml Cell Sciences CRG400BNeurotrophic Factor BDNF Brain-derived  20 ng/ml Cell Sciences CRB600BNeurotrophic Factor Shh Sonic Hedgehog, N-  50 ng/ml R&D 1845-SH-025terminal peptide RA Retinoic Acid  0.1 uM Sigma R2625 IGF-1 Insulin-likeGrowth  10 ng/ml PeproTech 100-11 Factor-I cAMP Adenosine 3′,5′-cyclic 1 uM Sigma A9501 Monophosphate CNTF Ciliary Neurotrophic  5 ng/ml CellSciences CRC400A Factor NT-3 Neurotrophin-3  20 ng/ml Cell SciencesCRN500B NT-4 Neurotrophin-4  20 ng/ml Cell Sciences CRN501B Vitronectin100 ng/ml Sigma V8379 Laminin Mouse Laminin  4 μg/ml Invitrogen23017-015 G5 (100X) 1X Invitrogen 17503-012 Agrin 100 ng/ml R&D550-AG-100

That which is claimed is:
 1. A method for forming at least one syntheticneuromuscular junction, the method comprising: co-culturingdifferentiated human skeletal muscle stem cells adhered to an artificialsurface and overlayered with differentiated human spinal cord stem cellsin a serum-free medium; and forming at least one functionalneuromuscular junction between a differentiated human skeletal musclestem cell and a differentiated human spinal cord stem cell.
 2. Themethod of claim 1, further comprising adding NbActiv4 to the serum-freemedium.
 3. The method of claim 1, wherein the artificial surfacecomprises a silicon based substrate monolayer deposited thereon.
 4. Themethod of claim 3, wherein the silicon based substrate monolayercomprises DETA.
 5. The method of claim 3, wherein the silicon basedsubstrate monolayer is deposited on the artificial surface in apredetermined pattern.
 6. A method of forming synthetic neuromuscularjunctions, the method comprising: adhering differentiated human skeletalmuscle stem cells to an artificial surface; overlayering differentiatedhuman spinal cord stem cells onto the differentiated human skeletalmuscle stem cells; culturing the artificial surface in a serum-freemedium, and monitoring for formation of functional neuromuscularjunctions.
 7. The method of claim 6, wherein the artificial surface iscoated with DETA.
 8. The method of claim 6, further comprising addingNbActiv4 to the serum-free medium.
 9. A co-culture for forming at leastone synthetic mammalian neuromuscular junction, comprising:differentiated human skeletal muscle stem cells adhered to an artificialsurface and overlayered with differentiated human spinal cord stem cellsin a serum-free medium.
 10. The co-culture of claim 9, wherein themedium comprises the components in Table
 1. 11. The co-culture of claim9, wherein the serum-free medium comprises at least one synaptogenesispromoting component and one or more trophic factors.
 12. The co-cultureof claim 9, wherein one or more of the differentiated human spinal cordstem cells is functionally linked to one or more of the differentiatedhuman skeletal muscle stem cells.
 13. The co-culture of claim 9, whereinthe artificial surface comprises a silicon based monolayer substratedeposited thereon.
 14. The co-culture of claim 13, wherein the siliconbased monolayer substrate is deposited in a predetermined pattern. 15.The co-culture of claim 13, wherein the silicon based monolayersubstrate comprises DETA.
 16. The co-culture of claim 9, wherein theserum-free medium further comprises NbActiv4.